Towards spruce-type photosystem II: consequences of the loss of light-harvesting proteins LHCB3 and LHCB6 in Arabidopsis.

The largest stable photosystem II (PSII) supercomplex in land plants (C2S2M2) consists of a core complex dimer (C2), two strongly (S2) and two moderately (M2) bound light-harvesting protein (LHCB) trimers attached to C2 via monomeric antenna proteins LHCB4-6. Recently, we have shown that LHCB3 and LHCB6, presumably essential for land plants, are missing in Norway spruce (Picea abies), which results in a unique structure of its C2S2M2 supercomplex. Here, we performed structure-function characterization of PSII supercomplexes in Arabidopsis (Arabidopsis thaliana) mutants lhcb3, lhcb6, and lhcb3 lhcb6 to examine the possibility of the formation of the "spruce-type" PSII supercomplex in angiosperms. Unlike in spruce, in Arabidopsis both LHCB3 and LHCB6 are necessary for stable binding of the M trimer to PSII core. The "spruce-type" PSII supercomplex was observed with low abundance only in the lhcb3 plants and its formation did not require the presence of LHCB4.3, the only LHCB4-type protein in spruce. Electron microscopy analysis of grana membranes revealed that the majority of PSII in lhcb6 and namely in lhcb3 lhcb6 mutants were arranged into C2S2 semi-crystalline arrays, some of which appeared to structurally restrict plastoquinone diffusion. Mutants without LHCB6 were characterized by fast induction of non-photochemical quenching and, on the contrary to the previous lhcb6 study, by only transient slowdown of electron transport between PSII and PSI. We hypothesize that these functional changes, associated with the arrangement of PSII into C2S2 arrays in thylakoids, may be important for the photoprotection of both PSI and PSII upon abrupt high-light exposure.

Spruce versus Arabidopsis: different strategies of photosynthetic acclimation to light intensity change.

The acclimation of higher plants to different light intensities is associated with a reorganization of the photosynthetic apparatus. These modifications, namely, changes in the amount of peripheral antenna (LHCII) of photosystem (PS) II and changes in PSII/PSI stoichiometry, typically lead to an altered chlorophyll (Chl) a/b ratio. However, our previous studies show that in spruce, this ratio is not affected by changes in growth light intensity. The evolutionary loss of PSII antenna proteins LHCB3 and LHCB6 in the Pinaceae family is another indication that the light acclimation strategy in spruce could be different. Here we show that, unlike Arabidopsis, spruce does not modify its PSII/PSI ratio and PSII antenna size to maximize its photosynthetic performance during light acclimation. Its large PSII antenna consists of many weakly bound LHCIIs, which form effective quenching centers, even at relatively low light. This, together with sensitive photosynthetic control on the level of cytochrome b6f complex (protecting PSI), is the crucial photoprotective mechanism in spruce. High-light acclimation of spruce involves the disruption of PSII macro-organization, reduction of the amount of both PSII and PSI core complexes, synthesis of stress proteins that bind released Chls, and formation of "locked-in" quenching centers from uncoupled LHCIIs. Such response has been previously observed in the evergreen angiosperm Monstera deliciosa exposed to high light. We suggest that, in contrast to annuals, shade-tolerant evergreen land plants have their own strategy to cope with light intensity changes and the hallmark of this strategy is a stable Chl a/b ratio.

Unique organization of photosystem II supercomplexes and megacomplexes in Norway spruce.

Photosystem II (PSII) complexes are organized into large supercomplexes with variable amounts of light-harvesting proteins (Lhcb). A typical PSII supercomplex in plants is formed by four trimers of Lhcb proteins (LHCII trimers), which are bound to the PSII core dimer via monomeric antenna proteins. However, the architecture of PSII supercomplexes in Norway spruce[Picea abies (L.) Karst.] is different, most likely due to a lack of two Lhcb proteins, Lhcb6 and Lhcb3. Interestingly, the spruce PSII supercomplex shares similar structural features with its counterpart in the green alga Chlamydomonas reinhardtii [Kouril et al. (2016) New Phytol. 210, 808-814]. Here we present a single-particle electron microscopy study of isolated PSII supercomplexes from Norway spruce that revealed binding of a variable amount of LHCII trimers to the PSII core dimer at positions that have never been observed in any other plant species so far. The largest spruce PSII supercomplex, which was found to bind eight LHCII trimers, is even larger than the current largest known PSII supercomplex from C. reinhardtii. We have also shown that the spruce PSII supercomplexes can form various types of PSII megacomplexes, which were also identified in intact grana membranes. Some of these large PSII supercomplexes and megacomplexes were identified also in Pinus sylvestris, another representative of the Pinaceae family. The structural variability and complexity of LHCII organization in Pinaceae seems to be related to the absence of Lhcb6 and Lhcb3 in this family, and may be beneficial for the optimization of light-harvesting under varying environmental conditions.

Organization of Plant Photosystem II and Photosystem I Supercomplexes.

In nature, plants are continuously exposed to varying environmental conditions. They have developed a wide range of adaptive mechanisms, which ensure their survival and maintenance of stable photosynthetic performance. Photosynthesis is delicately regulated at the level of the thylakoid membrane of chloroplasts and the regulatory mechanisms include a reversible formation of a large variety of specific protein-protein complexes, supercomplexes or even larger assemblies known as megacomplexes. Revealing their structures is crucial for better understanding of their function and relevance in photosynthesis. Here we focus our attention on the isolation and a structural characterization of various large protein supercomplexes and megacomplexes, which involve Photosystem II and Photosystem I, the key constituents of photosynthetic apparatus. The photosystems are often attached to other protein complexes in thylakoid membranes such as light harvesting complexes, cytochrome b 6 f complex, and NAD(P)H dehydrogenase. Structural models of individual supercomplexes and megacomplexes provide essential details of their architecture, which allow us to discuss their function as well as physiological significance.

Structural variability of plant photosystem II megacomplexes in thylakoid membranes.

Plant photosystem II (PSII) is organized into large supercomplexes with variable levels of membrane-bound light-harvesting proteins (LHCIIs). The largest stable form of the PSII supercomplex involves four LHCII trimers, which are specifically connected to the PSII core dimer via monomeric antenna proteins. The PSII supercomplexes can further interact in the thylakoid membrane, forming PSII megacomplexes. So far, only megacomplexes consisting of two PSII supercomplexes associated in parallel have been observed. Here we show that the forms of PSII megacomplexes can be much more variable. We performed single particle electron microscopy (EM) analysis of PSII megacomplexes isolated from Arabidopsis thaliana using clear-native polyacrylamide gel electrophoresis. Extensive image analysis of a large data set revealed that besides the known PSII megacomplexes, there are distinct groups of megacomplexes with non-parallel association of supercomplexes. In some of them, we have found additional LHCII trimers, which appear to stabilize the non-parallel assemblies. We also performed EM analysis of the PSII supercomplexes on the level of whole grana membranes and successfully identified several types of megacomplexes, including those with non-parallel supercomplexes, which strongly supports their natural origin. Our data demonstrate a remarkable ability of plant PSII to form various larger assemblies, which may control photochemical usage of absorbed light energy in plants in a changing environment.

Estimating heat tolerance of plants by ion leakage: a new method based on gradual heating.

Heat tolerance of plants related to cell membrane thermostability is commonly estimated via the measurement of ion leakage from plant segments after defined heat treatment. To compare heat tolerance of various plants, it is crucial to select suitable heating conditions. This selection is time-consuming and optimizing the conditions for all investigated plants may even be impossible. Another problem of the method is its tendency to overestimate basal heat tolerance. Here we present an improved ion leakage method, which does not suffer from these drawbacks. It is based on gradual heating of plant segments in a water bath or algal suspensions from room temperature up to 70-75°C. The electrical conductivity of the bath/suspension, which is measured continuously during heating, abruptly increases at a certain temperature TCOND (within 55-70°C). The TCOND value can be taken as a measure of cell membrane thermostability, representing the heat tolerance of plants/organisms. Higher TCOND corresponds to higher heat tolerance (basal or acquired) connected to higher thermostability of the cell membrane, as evidenced by the common ion leakage method. The new method also enables determination of the thermostability of photochemical reactions in photosynthetic samples via the simultaneous measurement of Chl fluorescence.

Light-induced gradual activation of photosystem II in dark-grown Norway spruce seedlings.

Gymnosperms, unlike angiosperms, are able to synthesize chlorophyll and form photosystems in complete darkness. Photosystem I (PSI) formed under such conditions is fully active, but photosystem II (PSII) is present in its latent form with inactive oxygen evolving complex (OEC). In this work we have studied light-induced gradual changes in PSII function in dark-grown cotyledons of Norway spruce (Picea abies) via the measurement of chlorophyll a fluorescence rise, absorption changes at 830 nm, thermoluminescence glow curves (TL) and protein analysis. The results indicate that in dark-grown cotyledons, alternative reductants were able to act as electron donors to PSII with inactive OEC. Illumination of cotyledons for 5 min led to partial activation of PSII, which was accompanied by detectable oxygen evolution, but still a substantial number of PSII centers remained in the so called PSII-Q(B)-non-reducing form. Interestingly, even 24 h long illumination was not sufficient for the full activation of PSII centers. This was evidenced by a weak attachment of PsbP protein and the absence of PsbQ protein in PSII particles, the absence of PSII supercomplexes, the suboptimal maximum yield of PSII photochemistry, the presence of C band in TL curve and also the presence of up-shifted Q band in TL in DCMU-treated cotyledons. This slow light-induced activation of PSII in dark-grown cotyledons could contribute to the prevention of PSII overexcitation before the light-induced increase in PSI/PSII ratio allows effective operation of linear electron flow.

Alternative electron transport mediated by flavodiiron proteins is operational in organisms from cyanobacteria up to gymnosperms.

Photo-reduction of O2 to water mediated by flavodiiron proteins (FDPs) represents a safety valve for the photosynthetic electron transport chain in fluctuating light. So far, the FDP-mediated O2 photo-reduction has been evidenced only in cyanobacteria and the moss Physcomitrella; however, a recent phylogenetic analysis of transcriptomes of photosynthetic organisms has also revealed the presence of FDP genes in several nonflowering plant groups. What remains to be clarified is whether the FDP-dependent O2 photo-reduction is actually operational in these organisms. We have established a simple method for the monitoring of FDP-mediated O2 photo-reduction, based on the measurement of redox kinetics of P700 (the electron donor of photosystem I) upon dark-to-light transition. The O2 photo-reduction is manifested as a fast re-oxidation of P700. The validity of the method was verified by experiments with transgenic organisms, namely FDP knock-out mutants of Synechocystis and Physcomitrella and transgenic Arabidopsis plants expressing FDPs from Physcomitrella. We observed the fast P700 re-oxidation in representatives of all green plant groups excluding angiosperms. Our results provide strong evidence that the FDP-mediated O2 photo-reduction is functional in all nonflowering green plant groups. This finding suggests a major change in the strategy of photosynthetic regulation during the evolution of angiosperms.

Transcriptional and post-translational control of chlorophyll biosynthesis by dark-operative protochlorophyllide oxidoreductase in Norway spruce.

Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7 °C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.

Evolutionary loss of light-harvesting proteins Lhcb6 and Lhcb3 in major land plant groups--break-up of current dogma.

Photosynthesis in plants and algae relies on the coordinated function of photosystems (PS) I and II. Their efficiency is augmented by finely-tuned light-harvesting proteins (Lhcs) connected to them. The most recent Lhcs (in evolutionary terms), Lhcb6 and Lhcb3, evolved during the transition of plants from water to land and have so far been considered to be an essential characteristic of land plants. We used single particle electron microscopy and sequence analysis to study architecture and composition of PSII supercomplex from Norway spruce and related species. We have found that there are major land plant families that lack functional lhcb6 and lhcb3 genes, which notably changes the organization of PSII supercomplexes. The Lhcb6 and Lhcb3 proteins have been lost in the gymnosperm genera Picea and Pinus (family Pinaceae) and Gnetum (Gnetales). We also revealed that the absence of these proteins in Norway spruce modifies the PSII supercomplex in such a way that it resembles its counterpart in the alga Chlamydomonas reinhardtii, an evolutionarily older organism. Our results break a deep-rooted concept of Lhcb6 and Lhcb3 proteins being the essential characteristic of land plants, and beg the question of what the evolutionary benefit of their loss could be.

Structural characterization of a plant photosystem I and NAD(P)H dehydrogenase supercomplex.

Cyclic electron transport (CET) around photosystem I (PSI) plays an important role in balancing the ATP/NADPH ratio and the photoprotection of plants. The NAD(P)H dehydrogenase complex (NDH) has a key function in one of the CET pathways. Current knowledge indicates that, in order to fulfill its role in CET, the NDH complex needs to be associated with PSI; however, until now there has been no direct structural information about such a supercomplex. Here we present structural data obtained for a plant PSI-NDH supercomplex. Electron microscopy analysis revealed that in this supercomplex two copies of PSI are attached to one NDH complex. A constructed pseudo-atomic model indicates asymmetric binding of two PSI complexes to NDH and suggests that the low-abundant Lhca5 and Lhca6 subunits mediate the binding of one of the PSI complexes to NDH. On the basis of our structural data, we propose a model of electron transport in the PSI-NDH supercomplex in which the association of PSI to NDH seems to be important for efficient trapping of reduced ferredoxin by NDH.

Cryo-EM structure of a plant photosystem II supercomplex with light-harvesting protein Lhcb8 and α-tocopherol.

The heart of oxygenic photosynthesis is the water-splitting photosystem II (PSII), which forms supercomplexes with a variable amount of peripheral trimeric light-harvesting complexes (LHCII). Our knowledge of the structure of green plant PSII supercomplex is based on findings obtained from several representatives of green algae and flowering plants; however, data from a non-flowering plant are currently missing. Here we report a cryo-electron microscopy structure of PSII supercomplex from spruce, a representative of non-flowering land plants, at 2.8 Å resolution. Compared with flowering plants, PSII supercomplex in spruce contains an additional Ycf12 subunit, Lhcb4 protein is replaced by Lhcb8, and trimeric LHCII is present as a homotrimer of Lhcb1. Unexpectedly, we have found α-tocopherol (α-Toc)/α-tocopherolquinone (α-TQ) at the boundary between the LHCII trimer and the inner antenna CP43. The molecule of α-Toc/α-TQ is located close to chlorophyll a614 of one of the Lhcb1 proteins and its chromanol/quinone head is exposed to the thylakoid lumen. The position of α-Toc in PSII supercomplex makes it an ideal candidate for the sensor of excessive light, as α-Toc can be oxidized to α-TQ by high-light-induced singlet oxygen at low lumenal pH. The molecule of α-TQ appears to shift slightly into the PSII supercomplex, which could trigger important structure-functional modifications in PSII supercomplex. Inspection of the previously reported cryo-electron microscopy maps of PSII supercomplexes indicates that α-Toc/α-TQ can be present at the same site also in PSII supercomplexes from flowering plants, but its identification in the previous studies has been hindered by insufficient resolution.

Heat-induced disassembly and degradation of chlorophyll-containing protein complexes in vivo.

Gradual heating of green leaves up to non-physiological temperatures is often used to estimate thermal stability of photosynthetic apparatus. However, a complete sequence of heat-induced disassembly and denaturation of chlorophyll-containing protein complexes (CPCs) has not been reported yet. In this work, we heated (1 degrees C x min(-1)) barley leaves to temperatures selected according to the changes in the chlorophyll fluorescence temperature curve (FTC) and we analyzed CPC stability by two-dimensional native Deriphat/SDS-PAGE. The first distinct change in both structure and function of photosystem II (PSII) appeared at 40-50 degrees C. PSII core (CCII) dimers began to dissociate monomers, which was accompanied by a decrease in PSII photochemistry and reflected in FTC as the first fluorescence increase. Further changes in CPCs appeared at 57-60 degrees C, when FTC increases to its second maximum. Photosystem I (PSI) cores (CCI) partially dissociated from light-harvesting complexes of PSI (LHCI) and formed aggregates. The rest of CCI-LHCI complexes, as well as the CCI aggregates, degraded to the PSI-A/B heterodimer in leaves heated to 70 degrees C. Heating to these temperatures led to a complete degradation of CCII components and corresponding loss of PSII photochemistry. Trimeric light-harvesting complexes of PSII (LHCII) markedly dissociated to monomers and denatured, as evidenced by a release of large amount of free chlorophylls. Between 70 and 80 degrees C, a complete degradation of LHCII occurred, leaving the PSI-A/B heterodimer as the only detectable CPC in the membrane. This most thermostable CPC disappeared after heating to 90 degrees C, which corresponded to a loss of PSI photochemistry.

Non-invasive monitoring of hydraulic surge propagation in a wounded tobacco plant.

BACKGROUND: When a plant is wounded, a rapid hydraulic surge, acting probably as a systemic signal, spreads from the site of injury throughout the plant and leads to small transient deformation of tissues. So far, the propagation of hydraulic surge has been monitored by contact and thus potentially invasive methods. RESULTS: Here we present a non-invasive optical method, which allows simultaneous monitoring of micrometric shift of two opposite stem margins. The usefulness of this method was demonstrated by the measurement of the hydraulic surge propagation in a tobacco (Nicotiana tabacum (L.) cv. Samsun) after burning of its upper leaf. We have observed transient narrowing the stem below the burned leaf, which started within a few minutes after local burning. The comparison of the shift of the stem margin following vascular trace of the burned leaf and the margin on the opposite side of the stem has revealed that the stem deformation is highly asymmetric. CONCLUSIONS: This optical method represents a novel tool to investigate the mechanism of systemic response of plants to local damage. Our results points out the complexity of the relationship between hydraulic surge propagation and stem deformation.

A dip in the chlorophyll fluorescence induction at 0.2-2 s in Trebouxia-possessing lichens reflects a fast reoxidation of photosystem I. A comparison with higher plants.

An unusual dip (compared to higher plant behaviour under comparable light conditions) in chlorophyll fluorescence induction (FI) at about 0.2-2 s was observed for thalli of several lichen species having Trebouxia species (the most common symbiotic green algae) as their native photobionts and for Trebouxia species cultured separately in nutrient solution. This dip appears after the usual O(J)IP transient at a wide range of excitation light intensities (100-1800 micromol photons m(-2) s(-1)). Simultaneous measurements of FI and 820-nm transmission kinetics (I(820)) with lichen thalli showed that the decreasing part of the fluorescence dip (0.2-0.4 s) is accompanied by a decrease of I(820), i.e., by a reoxidation of electron carriers at photosystem I (PSI), while the subsequent increasing part (0.4-2 s) of the dip is not paralleled by the change in I(820). These results were compared with that measured with pea leaves-representatives of higher plants. In pea, PSI started to reoxidize after 2-s excitation. The simultaneous measurements performed with thalli treated with methylviologen (MV), an efficient electron acceptor from PSI, revealed that the narrow P peak in FI of Trebouxia-possessing lichens (i.e., the I-P-dip phase) gradually disappeared with prolonged MV treatment. Thus, the P peak behaves in a similar way as in higher plants where it reflects a traffic jam of electrons induced by a transient block at the acceptor side of PSI. The increasing part of the dip in FI remained unaffected by the addition of MV. We have found that the fluorescence dip is insensitive to antimycin A, rotenone (inhibitors of cyclic electron flow around PSI), and propyl gallate (an inhibitor of plastid terminal oxidase). The 2-h treatment with 5 microM nigericin, an ionophore effectively dissipating the pH-gradient across the thylakoid membrane, did not lead to significant changes either in FI nor I(820) kinetics. On the basis of the presented results, we suggest that the decreasing part of the fluorescence dip in FI of Trebouxia-lichens reflects the activation of ferredoxin-NADP(+)-oxidoreductase or Mehler-peroxidase reaction leading to the fast reoxidation of electron carriers in thylakoid membranes. The increasing part of the dip probably reflects a transient reduction of plastoquinone (PQ) pool that is not associated with cyclic electron flow around PSI. Possible causes of this MV-insensitive PQ reduction are discussed.

Novel antitumor cisplatin and transplatin derivatives containing 1-methyl-7-azaindole: synthesis, characterization, and cellular responses.

The current work investigates the effect of new bifunctional and mononuclear Pt(II) compounds, the cis- and trans-isomers of [PtCl2(NH3)(L)] (L = 1-methyl-7-azaindole, compounds 1 and 2, respectively), on growth and viability of human carcinoma cells as well as their putative mechanism(s) of cytotoxicity. The results show that substitution of 1-methyl-7-azaindole for ammine in cisplatin or transplatin results in an increase of the toxic efficiency, selectivity for tumor cells in cisplatin-resistant cancer cells, and activation of the trans geometry. The differences in the cytotoxic activities of 1 and 2 were suggested to be due to their different DNA binding mode, different capability to induce cell cycle perturbations, and fundamentally different role of transcription factor p53 in their mechanism of action. Interestingly, both isomers make it possible to detect their cellular uptake and distribution in living cells by confocal microscopy without their modification with an optically active tag.

High-Temperature Induced Chlorophyll Fluorescence Rise in Plants at 40-50 degrees C: Experimental and Theoretical Approach.

We studied the temperature dependence of chlorophyll fluorescence intensity in barley leaves under weak and actinic light excitation during linear heating from room temperature to 50 degrees C. The heat-induced fluorescence rise usually appearing at around 40-50 degrees C under weak light excitation was also found in leaves treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or hydroxylamine (NH(2)OH). However, simultaneous treatment with both these compounds caused a disappearance of the fluorescence rise. We have suggested that the mechanism of the heat-induced fluorescence rise in DCMU-treated leaves is different than that in untreated or NH(2)OH-treated leaves. In DCMU-treated leaves, the heat-induced fluorescence rise reflects an accumulation of Q(A) (-) even under weak light excitation due to the thermal inhibition of the S(2)Q(A) (-) recombination as was further documented by a decrease in the intensity of the thermoluminescence Q band. Mathematical model simulating this experimental data also supports our interpretation. In the case of DCMU-untreated leaves, our model simulations suggest that the heat-induced fluorescence rise is caused by both the light-induced reduction of Q(A) and enhanced back electron transfer from Q(B) to Q(A). The simulations also revealed the importance of other processes occurring during the heat-induced fluorescence rise, which are discussed with respect to experimental data.

Origin of chlorophyll fluorescence in plants at 55-75 degrees C.

The origin of heat-induced chlorophyll fluorescence rise that appears at about 55-60 degrees C during linear heating of leaves, chloroplasts or thylakoids (especially with a reduced content of grana thylakoids) was studied. This fluorescence rise was earlier attributed to photosystem I (PSI) emission. Our data show that the fluorescence rise originates from chlorophyll a (Chl a) molecules released from chlorophyll-containing protein complexes denaturing at 55-60 degrees C. This conclusion results mainly from Chl a fluorescence lifetime measurements with barley leaves of different Chl a content and absorption and emission spectra measurements with barley leaves preheated to selected temperatures. These data, supported by measurements of liposomes with different Chl a/lipid ratios, suggest that the released Chl a is dissolved in lipids of thylakoid membranes and that with increasing Chl a content in the lipid phase, the released Chl a tends to form low-fluorescing aggregates. This is probably the reason for the suppressed fluorescence rise at 55-60 degrees C and the decreasing fluorescence course at 60-75 degrees C, which are observable during linear heating of plant material with a high Chl a/lipid ratio (e.g. green leaves, grana thylakoids, isolated PSII particles).